High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli

Jack D. Newman, Jessica Marshall, Michelle Chang, Farnaz Nowroozi, Eric Paradise, Douglas Pitera, Karyn L. Newman, Jay D. Keasling

Research output: Contribution to journalArticlepeer-review

228 Scopus citations

Abstract

Reconstructing synthetic metabolic pathways in microbes holds great promise for the production of pharmaceuticals in large-scale fermentations. By recreating biosynthetic pathways in bacteria, complex molecules traditionally harvested from scarce natural resources can be produced in microbial cultures. Here we report on a strain of Escherichia coli containing a heterologous, nine-gene biosynthetic pathway for the production of the terpene amorpha-4,11-diene, a precursor to the antimalarial drug artemisinin. Previous reports have underestimated the productivity of this strain due to the volatility of amorphadiene. Here we show that amorphadiene evaporates from a fermentor with a half-life of about 50 min. Using a condenser, we take advantage of this volatility by trapping the amorphadiene in the off-gas. Amorphadiene was positively identified using nuclear magnetic resonance spectroscopy and determined to be 89% pure as collected. We captured amorphadiene as it was produced in situ by employing a two-phase partitioning bioreactor with a dodecane organic phase. Using a previously characterized caryophyllene standard to calibrate amorphadiene production and capture, the concentration of amorphadiene produced was determined to be 0.5 g/L of culture medium. A standard of amorphadiene collected from the off-gas showed that the caryophyllene standard overestimated amorphadiene production by approximately 30%.

Original languageEnglish (US)
Pages (from-to)684-691
Number of pages8
JournalBiotechnology and Bioengineering
Volume95
Issue number4
DOIs
StatePublished - Nov 5 2006
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Keywords

  • Amorphadiene
  • Escherichia coli
  • Fermentation
  • Heterologous pathway
  • Terpene

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